Maturation of a sperm and an oocyte from their derivative stem cells involves ~400 and ~30 mitotic divisions, respectively, while there are ~1016 divisions per adult human lifetime. Chromosome segregation errors during division cause infertility, miscarriage, birth defects and cancer. Intimately linked mechanisms protect us from aneuploidy by fixing improper Microtubule-Kinetochore (MT-K) interactions (termed Error Correction) and by pausing the cell cycle (via the Spindle Checkpoint) before sister chromatid separation in anaphase. A central activity in both processes is Aurora B kinase. We have discovered a novel mechanistic link between Aurora B and Topoisomerase II (Topo II), an enzyme previously only known for its strand passage reaction, which alters DNA topology. It has been assumed that Topo II is essential for chromosome segregation due to its enzyme activity. However, an unanswered question is whether Topo II has additional functions at the kinetochores of chromosomes, where Topo II is most abundant in mitosis and where cell cycle checkpoint signals are generated. Using Xenopus egg extract assays, we found that SUMOylation of the Topo II C-terminal domain (CTD) does not alter enzyme activity but promotes specific binding of chromosomal proteins. Our discovery that CTD SUMOylation promotes the binding of cell cycle factors has revealed novel insight into the regulation of mitoti kinetochore function by Topo II. Among the proteins recruited to kinetochores by SUMOylated Topo II, are Claspin and Haspin which regulate the spindle checkpoint kinase Aurora B. Based on this finding, our central hypothesis is that Topo II-mediated regulation of Aurora B via Claspin and Haspin plays a critical role in checkpoint regulation during mitosis and that this mechanism is conserved in eukaryotes. We will examine this by combining yeast genetics, Xenopus egg extract cell-free assays, and genetically modified human cell lines. We will manipulate the SUMOylation status of Topo II to control interaction with Claspin and Haspin. We will elucidate: 1) The role of Topo II-SUMOylation in regulation of Aurora B activity via Claspin and Haspin. 2) The role of Topo II-dependent regulation of Aurora B in mitotic checkpoint signaling. These studies will define a novel regulatory step that controls kinetochore function as a molecular generator of mitotic checkpoint signals. The proposed research will uncover the novel function of kinetochore localized Topo II as a signaling molecule for mitotic checkpoint regulation. Elucidating the molecular mechanism of this signaling function of Topo II will allow the development of improved Topo II targeted therapeutics for cancer treatment.